Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science, National Institute for Natural Sciences, 38 Nishigo-Naka, Myodaiji, Okazaki, 444-8585, Japan.
Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), 38 Nishigo-Naka, Myodaiji, Okazaki, 444-8585, Japan.
Sci Rep. 2018 Jun 11;8(1):8803. doi: 10.1038/s41598-018-27131-8.
KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyclic phosphorylation and dephosphorylation at Ser431 and Thr432 in the C2 domain proceed with a period of approximately 24 h in the presence of other clock proteins, KaiA and KaiB, but recent studies have revealed a crucial role for the C1 ring in determining the cycle period. In this study, we mapped dynamic structural changes of the C1 ring in solution using a combination of site-directed tryptophan mutagenesis and fluorescence spectroscopy. We found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure. This transition triggered by ATP hydrolysis in the C1 ring in specific phosphorylation states is a necessary event for recruitment of KaiB, limiting the overall rate of slow complex formation. Our results provide structural and kinetic insights into the C1-ring rearrangements governing the slow dynamics of the cyanobacterial circadian clock.
KaiC 是蓝藻生物钟的核心振荡器，由 N 端 C1 结构域和 C 端 C2 结构域组成，在结合 ATP 后组装成双环六聚体。在其他生物钟蛋白 KaiA 和 KaiB 的存在下，C2 结构域中 Ser431 和 Thr432 的周期性磷酸化和去磷酸化过程大约每 24 小时进行一次，但最近的研究揭示了 C1 环在确定周期中的关键作用。在这项研究中，我们使用定点色氨酸突变和荧光光谱学相结合的方法，在溶液中绘制了 C1 环的动态结构变化图谱。我们发现，C1 环在保持其六聚体环结构的同时，伴随着 ATP 酶活性和磷酸化状态发生结构转变。这种由特定磷酸化状态下 C1 环中的 ATP 水解触发的转变是 KaiB 募集的必要事件，限制了慢复合物形成的整体速率。我们的结果为控制蓝藻生物钟慢动力学的 C1 环重排提供了结构和动力学见解。
